以肽脱甲酰基酶为靶点的抗结核药物高通量筛选模型的建立和应用

目的建立以肽脱甲酰基酶(PDF)为靶点的抗结核药物高通量筛选模型,应用该模型筛选得到活性微生物发酵液粗提物样品。方法以结核分枝杆菌H37Rv基因组为模板,扩增肽脱甲酰基酶的基因片段def,构建表达载体pET-28a-def,表达并纯化结核分枝杆菌PDF酶;基于PDF水解三肽底物for-Met-Ala-Ser释放出游离NH2,而游离NH2可与荧光胺反应产生荧光的原理,利用测定所产生荧光值的方法,建立高通量药物筛选模型;使用该模型对12400个微生物发酵液粗提物样品进行筛选,同时以耻垢分枝杆菌为检定菌,平板纸片法检测样品的抗菌活性,并检测所得阳性样品的细胞毒性。结果成功构建了表达载体pET-28a-def;所建立的模型稳定可行,可用于以肽脱甲酰基酶为靶点的抗结核药物的高通量筛选;用该模型对12400个微生物发酵液粗提物样品进行筛选,最终得到8个对肽脱甲酰基酶抑制活性和抗耻垢分枝杆菌活性均较好的阳性样品,阳性率0.06%;其中5个样品的细胞毒性较小。结论建立了灵敏度好、稳定性高的结核分枝杆菌肽脱甲酰基酶抑制剂高通量药物筛选模型,应用该模型所得到的阳性样品具有进一步深入研究的意义。     

人胚胎主动脉血管内皮祖细胞的分离、培养及鉴定

目的研究人胚胎血管内皮祖细胞(EPCs)分离、扩增及鉴定的方法,并评价其分化成血管内皮细胞的能力,为人胚胎血管来源EPCs作为干细胞技术治疗疾病的细胞材料提供依据。方法从14周龄流产人胚胎主动脉中应用胶原酶消化法分离获得EPCs,用含有碱性成纤维细胞生长因子、表皮生长因子和白血病抑制因子的低血清培养基体外扩增培养EPCs。分离培养的EPCs鉴定采用细胞免疫荧光染色、RT-PCR及流式细胞术,检测EPCs细胞的特异标志CD133、CD34和血管内皮细胞生长因子受体2(VEGFR2)。培养的EPCs应用VEGF进行诱导分化,并评价其分化成为血管内皮细胞的能力。结果分离的人胚胎主动脉EPCs细胞表达EPCs的标志分子CD133、CD34和VEGFR2。EPCs在体外特定低血清培养条件下表现很强的增殖能力。培养的EPCs细胞经过VEGF诱导后,细胞表达CD133明显降低,表达vWF、CD31和ELAM-1增强,并且体外成管能力和摄取Ac-LDL能力增强,表明细胞分化成为血管内皮细胞。结论人胚胎早期主动脉的EPCs具有很好的体外自我更新能力和分化成为血管内皮细胞的潜能,可作为EPCs治疗疾病的细胞材料。     

携带miR29c重组腺病毒的制备及其促骨髓瘤细胞凋亡作用

目的制备携带miR29c的重组腺病毒Ad5F11p-miR29c,评价miR29c对骨髓瘤细胞凋亡的影响。方法巢式PCR扩增得到hsa-miR29c,将hsa-miR29c基因克隆到腺病毒穿梭质粒pShuttle-CMV,得到pShuttle-CMV-miR29c。鉴定正确后,PmeI线性化并转化含Ad5F11p骨架质粒的BJ5183感受态细胞,重组获得腺病毒载体。PacI线性化的重组腺病毒质粒转染HEK293细胞,制备重组腺病毒Ad5F11p-miR29c。以对照病毒Ad5F11p-EGFP感染人骨髓瘤细胞系SKO-007、U266和XG7,通过流式细胞术确定最佳感染复数。Ad5F11p-miR29c以最佳感染复数感染骨髓瘤细胞,流式细胞术检测miR29c对细胞凋亡的影响。结果 PCR扩增获得miR29c基因,成功将其连接到腺病毒穿梭质粒pShuttle-CMV,经测序鉴定正确。采用Ad-easy系统,成功构建并制备了CMV启动的重组腺病毒Ad5F11p-miR29c。流式细胞术结果显示,Ad5F11p-EGFP对SKO-007和U266细胞的最佳感染复数为150MOI;而XG7为100MOI。Ad5F11p-miR29c以最佳感染复数感染细胞48h后,细胞凋亡率升高。SKO-007细胞的凋亡率达到26.5%,XG7细胞的凋亡率达到46.9%。结论成功制备重组腺病毒Ad5F11p-miR29c,并证实miR29c能够诱导骨髓瘤细胞凋亡。     

LC-MS/MS测定大鼠血浆中黄藤素含量及其药代动力学研究

目的建立大鼠血浆中黄藤素的LC-MS/MS定量方法,并利用此方法考察黄藤素在大鼠体内的药物动力学。方法色谱柱为ZorbaxEclipseXDB-C1(82.1mm×50mm,1.8μm),流动相为0.1%甲酸溶液-乙腈(70:30),采用多级反应模式(MRM)检测;通过大鼠眼底静脉取血,血浆经乙腈沉淀蛋白,取上清液进行LC-MS/MS分析,测定黄藤素血药浓度,通过DAS2.0计算其药动学参数。结果血浆中黄藤素在0.442~44.2ng/ml范围内呈现良好的线性关系,方法学精密度、回收率、稳定性等均符合要求;大鼠体内黄藤素药动学符合二室模型特征,灌胃给予40mg/kg黄藤素后,大鼠血浆中的黄藤素浓度在2.8h达峰值,t1/2为6h,最大血药浓度为19.8ng/ml。结论本研究建立的大鼠体内黄藤素测定方法灵敏度高、专一性好,可用于黄藤素的体内药物动力学研究,为黄藤素应用研究提供了有力支持。     

红树林植物内生放线菌I07A-01824发酵液中杀线虫活性成分的分离、纯化与鉴定

目的建立稳定有效的体外杀线虫模型,并从红树林植物木榄内生放线菌I07A-01824的发酵液中分离纯化具有杀线虫活性的物质,并鉴定其化学结构。方法在体外杀线虫模型指导下,利用硅胶层析、高效液相色谱等方法对放线菌I07A-01824的发酵液进行分离纯化,得到高纯度的杀线虫活性物质;通过现代波谱分析技术对其进行结构解析,确定其化学结构。结果红树林内生放线菌I07A-01824的次级代谢产物中具有较强杀线虫活性的物质,为一不饱和脂肪酸,化学结构为5,8-二烯十四酸;27.3mg/L剂量下开始显示对秀丽隐杆线虫有活性,其24h后的LC50值为162.8mg/L。结论此不饱和脂肪酸对线虫有较强的致死作用,并且在内生放线菌I07A-01824菌株的次级代谢产物中稳定存在。     

链霉菌I06A-00625发酵产物的提取分离、结构鉴定及活性研究

目的从链霉菌I06A-00625(cpcc201504)的代谢产物中分离得到具有抑菌活性的抗生素,并进行结构鉴定。方法通过大孔吸附树脂X-5、葡聚糖凝胶LH-20和HPLC色谱对发酵液中活性成分进行分离纯化;根据紫外、高分辨质谱并结合1H-NMR和13C-NMR数据确定化合物结构,并进行抗菌活性研究。结果分离纯化得到6个单一组分,分别为化合物I06A625A、206A和206B及3个四烯大环内酯类抗生素Tetramycin A、Tetramycin B和Tetrin A。其中206A为首次从天然产物中分离得到。I06A625A具有抗铜绿色假单胞菌活性;Tetramycin A、Tetramycin B和Tetrin A具有抗真菌活性。结论链霉菌I06A-00625可产生抗铜绿色假单胞菌的核苷类化合物及抗真菌的四烯类化合物等多种活性成分。     

医用自交联透明质酸钠凝胶预防兔硬膜外粘连的安全性研究

目的利用体感诱发电位及运动功能评分,评价医用自交联透明质酸钠凝胶预防椎板切除术后硬膜外粘连的安全性。方法将L5全椎板切除的新西兰兔24只随机分为4组,A组为对照组:术区生理盐水冲洗后关闭切口;B组、C组、D组均为实验组:均在术区硬脊膜暴露区覆盖医用自交联透明质酸钠凝胶,而覆盖剂量各不相同,分别为0.5、0.75和1.0ml。分别于麻醉后,椎板切除术后,闭合手术切口之后,术后8周这4个时间点进行体感诱发电位监测并记录潜伏期值。并分别于术前,术后1d,术后8周对各组动物进行后肢运动功能评分。结果麻醉后和椎板切除术后各组潜伏期值无统计学差异(P0.05)。在关闭切口后测定各组的潜伏期值显示:A组和B组潜伏期值均无延迟(P0.05),C组和D组潜伏期值出现明显延迟(P0.05)。在术后8周复测各组潜伏期值,均处于正常范围(P0.05)。运动功能评分结果:术前所有动物评分均正常。在术后1d,A组和B组动物的评分仍然正常,C组和D组的动物评分下降。在术后8周,各组动物的运动功能评分均再次正常。结论体感诱发电位是测定脊髓损伤的敏感指标;椎板切除术后局部覆盖预防粘连剂有剂量的要求,0.5ml的医用自交联透明质酸钠凝胶不会对该动物模型的脊髓的电生理功能及后肢运动功能产生影响。     

骨形态发生蛋白9体外调节乳腺癌细胞与骨髓间充质干细胞的相互作用

 目的 观察骨形态发生蛋白9（BMP9）在共培养条件下对HS-5成骨分化及MDA-MB-231转移相关因子表达的影响。方法 采用Transwell小室间接共培养MDA-MB-231细胞与HS-5细胞,加入BMP9条件培养基,使用化学发光法、细胞化学染色法及茜素红S染色法分别检测HS-5细胞碱性磷酸酶（ALP）活性、ALP表达及细胞钙盐沉积的变化;应用RT-PCR及Western blot观察HS-5细胞骨钙素（OCN）、MDA-MB-231细胞甲状旁腺激素相关蛋白（PTH-rP）及白细胞介素6(IL-6)mRNA和蛋白表达的变化。结果 HS-5的ALP活性升高,并呈时间依赖性,第9天达最高,随后降低;ALP细胞化学染色结果进一步证实这一点;HS-5细胞钙盐沉积增多、OCN mRNA和蛋白的表达升高（P<0.05）;MDA-MB-231转移相关的PTH-rP及IL-6 mRNA和蛋白的表达下降（P<0.05）。结论 BMP9能够调节乳腺癌细胞与骨髓间充质干细胞的相互作用。     

肝原位移植瘤裸鼠模型的建立及活体荧光成像检测

目的 建立能够通过活体荧光成像系统实时监测肿瘤进展的肝原位移植瘤裸鼠模型.方法 重组质粒pcDNA3.1-Luc转染细胞构建稳定表达荧光素酶的PLC/PRF/5肝癌细胞株,将该细胞注入裸鼠肝脏实质内,应用活体成像技术动态监测肿瘤进展情况,解剖荧光信号阳性裸鼠观察肿瘤组织生长情况.免疫荧光检测肿瘤组织中HBsAg表达.结果 对裸鼠肝原位移植瘤应用活体荧光系统成像,在肿瘤细胞注射部位检测到荧光信号,解剖动物后可在肝脏组织中观察到异质细胞团块.免疫荧光证实移植瘤中有HBsAg表达.结论 肝脏原位移植瘤裸鼠模型建立成功,为抗肝癌药物的研发提供了工具.     

活体动物光学成像技术与应用研究

活体动物光学成像是利用生物发光及荧光技术在活体动物体内进行生物标记通过光学成像系统来监测被标记动物体内分子及细胞等的生物学过程。按发光模式可分为生物发光和荧光两类。相对于传统动物实验研究方法,具有无创、可多次重复、实时活体成像、灵敏、安全等优势,这项技术在标记活体内肿瘤活体细胞示踪、标记基因及转基因动物等方面的应用广泛。     

内皮抑素基因转移对乳腺癌细胞生长影响的体内和离体研究

目的：探讨内皮抑素(ES)基因转移在乳腺癌抗血管新生中的作用。 方法: 通过建立逆转录病毒介导的ES基因转移系统，用ES病毒转染人乳腺癌细胞系MDA-MB-231。以聚合酶链反应（PCR）、MTT法和裸鼠成瘤实验分析ES的生物学特性及其功能。 结果： 脂质体转染与交互感染策略获得ES病毒生产细胞；以ES病毒转染MDA-MB-231细胞后，经PCR分析显示其内有ES基因整合并持续表达，其分泌的ES能明显抑制内皮细胞EA.hy926的增殖(P<0.05)，但对肿瘤细胞的离体生长无明显影响（P>0.05）；裸鼠皮下移植瘤模型表明，ES基因表达可明显抑制MDA-MB-231细胞的生长(P<0.01)；实验组的肿瘤微血管密度（MVD）和血管内皮生长因子(VEGF)表达低于对照组(P<0.05)。 结论: 逆转录病毒载体介导的ES基因在乳腺癌细胞中可有效表达，能明显抑制血管内皮细胞生长，并通过旁分泌方式抑制血管新生，具有显著的抗肿瘤生长的作用。     

Urokinase-Receptor/Integrin Complexes Are Functionally Involved in Adhesion and Progression of Human Breast Cancer in Vivo

Interactions between specific cell-surface molecules, which include the urokinase receptor (uPAR) and integrins, are crucial to processes of tumor invasion and metastasis. Here we demonstrate that uPAR and beta1-integrins may cluster at distinct sites at the cell surface of metastatic MDA-MB-231 breast cancer cells and form functional complexes. Attachment assays performed in the presence of a synthetic peptide (p25), which interferes with the formation of uPAR-integrin complexes, reveal that uPAR is able to regulate the adhesive function of integrins in breast cancer cells. On dissociation of the uPAR-integrin complexes by p25, tumor cell attachment to the extracellular matrix was either decreased (vitronectin) or increased (fibronectin). Moreover, the tumor cells display remarkable morphological changes when cultured on fibronectin in the continuous presence of p25, leading to increased cell spreading and attachment. In marked contrast to control conditions, increased cellular adhesion to fibronectin after p25 treatment was entirely beta1-integrin-mediated. The role of uPAR-integrin complexes in tumor progression was studied in an in vivo bone xenograft model. Stably transfected MDA-MB-231 cells that overexpress p25 showed a significant reduction in tumor progression in bone (P < or = 0.0001 versus mock-control). In line with these observations, continuous administration of p25 (25 microg/mouse/day, osmotic minipumps) for 28 days resulted in significantly reduced tumor progression of MDA-MB-231 cells in bone (P < or = 0.005) when compared to scrambled control peptide. In conclusion, our data demonstrate that uPAR can act as an adhesion receptor in breast cancer and is capable of regulating integrin function. Our findings strongly suggest that adhesive and proteolytic events are tightly associated in metastatic breast cancer cells and that functional integrin-uPAR complexes are involved in tumor progression in vivo.     

Localization of osteoblast inflammatory cytokines MCP-1 and VEGF to the matrix of the trabecula of the femur,a target area for metastatic breast cancer cell colonization

Bone likely provides a hospitable environment for cancer cells as suggested by their preferential localization to the skeleton. Previous work has shown that osteoblast-derived cytokines increased in the presence of metastatic breast cancer cells. Thus, we hypothesized that osteoblast-derived cytokines, in particular IL-6, MCP-1, and VEGF, would be localized to the bone metaphyses, an area to which breast cancer cells preferentially traffic. Human metastatic MDA-MB-231 breast cancer cells were inoculated into the left ventricle of the heart of athymic mice. Three to four weeks later, tumor localization within isolated femurs was examined using μCT and MRI. In addition, IL-6, MCP-1, and VEGF localization were assayed via immunohistochemistry. We found that MDA-MB-231 cells colonized trabecular bone, the area in which murine MCP-1 and VEGF were visualized in the bone matrix. In contrast, IL-6 was expressed by murine cells throughout the bone marrow. MDA-MB-231 cells produced VEGF, whose expression was not only associated with the breast cancer cells, but also increased with tumor growth. This is the first study to localize MCP-1, VEGF, and IL-6 in bone compartments via immunohistochemistry. These data suggest that metastatic cancer cells may co-opt bone cells into creating a niche facilitating cancer cell colonization.     

Molecular imaging for assessment of mesenchymal stem cells mediated breast cancer therapy

The tumor tropism of mesenchymal stem cells (MSCs) makes them an excellent delivery vehicle used in anticancer therapy. However, the exact mechanisms of MSCs involved in tumor microenvironment are still not well defined. Molecular imaging technologies with the versatility in monitoring the therapeutic effects, as well as basic molecular and cellular processes in real time, offer tangible options to better guide MSCs mediated cancer therapy. In this study, an in situ breast cancer model was developed with MDA-MB-231 cells carrying a reporter system encoding a double fusion (DF) reporter gene consisting of firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP). In mice breast cancer model, we injected human umbilical cord-derived MSCs (hUC-MSCs) armed with a triple fusion (TF) gene containing the herpes simplex virus truncated thymidine kinase (HSV-ttk), renilla luciferase (Rluc) and red fluorescent protein (RFP) into tumor on day 13, 18, 23 after MDA-MB-231 cells injection. Bioluminescence imaging of Fluc and Rluc provided the real time monitor of tumor cells and hUC-MSCs simultaneously. We found that tumors were significantly inhibited by hUC-MSCs administration, and this effect was enhanced by ganciclovir (GCV) application. To further demonstrate the effect of hUC-MSCs on tumor cells in vivo, we employed the near infrared (NIR) imaging and the results showed that hUC-MSCs could inhibit tumor angiogenesis and increased apoptosis to a certain degree. In conclusion, hUC-MSCs can inhibit breast cancer progression by inducing tumor cell death and suppressing angiogenesis. Moreover, molecular imaging is an invaluable tool in tracking cell delivery and tumor response to hUC-MSCs therapies as well as cellular and molecular processes in tumor.     

The regulatory role of Annexin 3 in a nude mouse bearing a subcutaneous xenograft of MDA-MB-231 human breast carcinoma

The following study investigated the effects of Annexin A3 (ANXA3) on breast cancer biological behavior in vivo, using nude mouse model bearing a subcutaneous tumor. A total of 18 female nude mice were randomly divided into three groups (n?=?6): negative control group which was inoculated with MDA-MB-231 cells, blank control group which was inoculated with MDA-MB-231-NC cells, and the transfection group which was inoculated with MDA-MB-231-Sh cells. The experiment lasted for 4 weeks, during which mice conditions, diet and defecation were monitored on a daily basis. Body weight, as well as tumor diameters, which were assessed using standard caliper method, were measured once a week. In vivo imaging was performed to detect the activity of transplanted tumors. H&E staining was used to analyze the histological structure of tumor tissues in three groups, while flow cytometry and fluorescent RT-PCR were performed to measure cell proliferation and the expression of ANXA3 mRNA. Briefly, significantly slower tumor growth and tumor activity were observed in the transfection group compared to negative and blank controls, while the tumor weight and volume in this group were also significantly lower compared to the other two groups (P < 0.01). Sparse tumor cells accompanied with massive fibrous connective tissue proliferation, and lower new blood vessels formation were observed in transfection group compared to other groups. Moreover, mRNA and protein levels of ANXA3 were significantly lower in transfection group compared to the other two groups (P < 0.01). In addition, lower proliferation index and higher G_0/1 cell count were observed in transfection group compared to negative and blank controls (P < 0.01). To sum up, these results suggested that ANXA3 silencing regulates the proliferation and inhibits the growth of MDA-MB-231 breast cancer cells. Consequently, ANXA3 might be used as a potential target for gene therapy in breast cancer.     

Combined treatment with paclitaxel and suramin prevents the development of metastasis by inhibiting metastatic colonization of circulating tumor cells

Metastatic disease accounts for most deaths due to breast cancer and thus identification of novel ways to prevent this complication remains a key goal. A frequently employed preclinical model of breast cancer metastasis relies on xenografted human MDA-MB-231 cells, since these reliably produce both soft tissue and osseous metastases when introduced into the arterial circulation of athymic mice. Herein, we explored the ability of suramin (SA), an agent shown to antagonize the effects of various stromal cell-derived growth factors relevant to bone marrow colonization of tumor cells, administered both with and without paclitaxel (PTX), to inhibit the development of MDA-MB-231 metastases. Treatment with SA, PTX, or PTX plus SA (PTX/SA) was begun either at day-1, or 7 days after intra-arterial inoculation of luciferase-expressing MDA-MB-231-luc2 cells. Using in vivo and ex vivo bioluminescence imaging to detect macro-metastases, we found that PTX/SA treatment initiated on day-1 was able to dramatically reduce the frequency of bone metastases. PTX/SA and PTX administration commenced at day 7, in contrast, had no significant effect on the frequency of bone metastases, but exerted a relatively modest inhibitory effect on growth of metastases. Interestingly, reminiscent of what is seen clinically in anti-HER2 treated individuals, several of the PTX/SA-treated long term survivors went on to develop late onset CNS metastasis. Our results suggest that combining SA with PTX either in an adjuvant setting or during medical interventions that can increase the numbers of circulating tumour cells might be an effective way to prevent the development of metastases.     

Theranostic vitamin E TPGS micelles of transferrin conjugation for targeted co-delivery of docetaxel and ultra bright gold nanoclusters

The aim of this work was to develop an advanced theranostic micelles of D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), which are conjugated with transferrin for targeted co-delivery of docetaxel (DTX) as a model drug and ultra bright gold clusters (AuNC) as a model imaging agent for simultaneous cancer imaging and therapy. The theranostic micelles with and without transferrin conjugation were prepared by the solvent casting method and characterized for their particle size, polydispersity, surface chemistry, drug encapsulation efficiency, drug loading and cellular uptake efficiency. Transferrin receptors expressing MDA-MB-231-luc breast cancer cells and NIH-3T3 fibroblast cells (control cells without transferrin receptor expression) were employed as an in vitro model to access cytotoxicity of the formulations. The overexpression of transferrin receptor on the surface of MDA-MB-231-luc cells was confirmed by flow cytometry. The biodistribution study and theranostic efficacy of the micelles were investigated by using the Xenogen IVIS^® Spectrum imaging system, which includes AuNC based fluorescence imaging and luciferase induced bioluminescence imaging on MDA-MB-231-luc tumor bearing SCID mice. The IC₅₀ values demonstrated that the non-targeted and targeted micelles could be 15.31 and 71.73 folds more effective than Taxotere^® after 24 h treatment with the MDA-MB-231-luc cells. Transferrin receptor targeted delivery of such micelles was imaged in xenograft model and showed their great advantages for real-time tumor imaging and inhibition of tumor growth.     

乳腺癌微环境成纤维细胞对乳腺癌细胞表达TIGAR 和Bcl-2 的影响

目的:探索乳腺癌微环境成纤维细胞对乳腺癌细胞表达TIGAR 和Bcl-2 的影响及对乳腺癌生长的作用。方法:体外实验,建立了人乳腺癌细胞株MDA-MB-231 和人成纤维细胞株CCC-ESF-1 共培养模型,RT-qPCR 和Western blot 检测成纤维细胞对乳腺癌细胞表达TIGAR 和Bcl-2 的影响,Annexin V 流式细胞术和Caspase-3 活性荧光检测乳腺癌细胞的凋亡;体内实验,建立人乳腺癌荷瘤裸鼠模型,测量荷瘤裸鼠肿瘤体积,免疫组化检测移植瘤组织TIGAR 和Bcl-2 的表达。结果:体外实验研究结果显示,共培养的成纤维细胞可上调MDA-MB-231 细胞TIGAR 和Bcl-2 的表达并抑制MDA-MB-231 细胞的凋亡;体内实验研究结果显示,与乳腺癌细胞共植入的成纤维细胞能上调荷瘤裸鼠乳腺癌组织TIGAR 和Bcl-2 的表达,高表达的TIGAR 和Bcl-2 可加速荷瘤裸鼠乳腺癌组织生长。结论:乳腺癌微环境成纤维细胞可上调乳腺癌细胞TIGAR 和Bcl-2 的表达,高表达的TIGAR 和Bcl-2 抑制乳腺癌细胞的凋亡,促进了乳腺癌的生长。     

Curcumin induces apoptosis in breast cancer cells and inhibits tumor growth in vitro and in vivo

Curcumin has shown therapeutic and/or adjuvant therapeutic effects on the treatment of some patients with breast cancer. However, its mechanisms of action are largely unknown. This study was designed to investigate its antitumor effect and underlying mechanisms in human breast cancer MDA-MB-231 and MCF-7 cells. The MTT assay was used to evaluate cell viability, and flow cytometry, acridine orange staining and transmission electron microscopy were used to detect apoptosis for cultured cells. The protein expression in cells was evaluated by western blot analysis. Breast tumors were established by subcutaneous injection of MDA-MB-231 cells in nude BALB/c mice, and curcumin was administered to the mice. The size of tumors was monitored and the weight of tumors was examined. The exposure of breast cancer cells to curcumin resulted in growth inhibition and the induction of apoptosis in a dose-dependent manner. We also found that the expression of Bcl-2 protein decreased and the expression of Bax protein increased which lead to an increase of the Bax/Bcl-2 ratio. In mice bearing MDA-MB-231 xenograft tumors, administration of curcumin showed a significant decrease of tumor volumes and tumor weight compared with the control. Our results showed that curcumin exhibited antitumor effects in breast cancer cells with an induction of apoptosis.     

Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.